The present invention relates to a method for treating microbial infections of mammals, including humans and other primates; a method for killing bacteria and fungi; and a method for treating material subject to microbial contamination by administration of an effective antimicrobial amount of reptilian hemoglobin, or of the xcex1 or xcex2 chains of this molecule, free of heme, fragments therefrom and combinations thereof. The invention also relates to compositions comprising such proteins, polypeptides or fragments.
Antibacterial peptides from natural sources have a long history. In 1939 Dubos demonstrated that a soil bacillus, subsequently identified as B. brevis, produced substances that could prevent pneumococcal infections in mice. Subsequently, Hotchkiss and Dubos purified two substances composed of amino acids and one of these, gramicidin, became available as a therapeutic agent. Subsequent studies on antimicrobial peptides have identified many active agents (1). (Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references, listed in sequence, may be found at the end of the specification. All of these references and any additional references cited within this application are herein incorporated by reference in their entirety.)
Many bacteria produce antimicrobial peptides (bacteriocins) and proteins; those released from Gram-negative bacteria are the more potent and have the wider spectrum of activity (2). The defensins are small antimicrobial peptides found in neutrophils, non-human macrophages and Paneth cells (3). Amphibian skin is a rich source of antimicrobial peptides, one of these, magainin, isolated from Xenopus laevis, currently is undergoing clinical trial (4,5). Plants form a variety of gene-encoded antimicrobial peptides including the phytoalexins, the PR proteins and the AMPs (6,7). Insects have been shown to synthesize bacteriocidal peptides and proteins such as cecropin obtained from the moth Cecropia (8,9,10) and the sarcotoxins obtained from the larvae of the flesh fly Sarcocphaga perigrina (11). The hemocytes of the horse-shoc crab Limulus are the source of the tachyplesins and squalamine, an aminosteroid with antimicrobial activity, has been isolated from the shark, Squalus acanthias (12).
Thus, many antimicrobial substances lie within the families of xe2x80x9cnaturalxe2x80x9d antibiotics such as the cecropins, magainins, defensins, serprocidins and others. These substances are widely distributed in nature and provide an innate defense mechanism against infection in species ranging from insects to amphibians to mammals. Generally these substances are stored in cells, to be induced and secreted within the animal when challenged. Many act by disrupting the bacterial cell membrane selectively; many would be toxic to host cells as well, were they not sequestered (13). A number of these compounds have been proposed as being useful as antimicrobial agents (14,15).
Hemoglobin (MW=64,500) consists of four polypeptide chains and four heme prosthetic groups in which the iron atoms are in the ferrous state. The protein, called globin, consists of two xcex1 chains and two xcex2 chains. In the alligator Alligator mississippiensis, the xcex1 chain contains 141 amino acid residues and the xcex2 chain contains 146 residues. The amino acid sequence of the xcex1 (SEQ ID NO:2) and xcex2 (SEQ ID NO:1) chains of alligator hemoglobin, is a follows:
The structure of heme (ferroprotoporphyrin IX) is well known.
One heme group is bound to each polypeptide chain through a coordination bond between the iron atom and the R group of a histidine residue. The sixth coordination bond of the iron atom is available to bind oxygen. In addition, hemoglobin also transports H+, CO2, and NO. The structure of heme is identical in all animals that have hemoglobin but the sequence of the globin chains varies considerably. In spite of this variation, the configuration of the tetramer is quite similar among species.
The interactions of hemoglobin with oxygen and carbon dioxide depend on the state of the heme and the residues surrounding it, as well as on regulation by heterotrophic ligands including H+, Clxe2x88x92, CO2 , HCO3xe2x88x92 and 2,3,diphosphoglycerate. These ligands regulate the equilibrium between the high affinity state (the relaxed or R structure), and the low affinity tense state (or T structure). The stereochemistry of hemoglobin has been reviewed extensively (16,17).
The hemoglobin of reptiles, Chelonia (turtles), Crocodilia (crocodilians) and Squamata (snakes and lizards) shows certain unique structural characteristics (18). In several species the hemoglobin tetramers have been found to form disulfide bridges with each other (19), although this may be largely an in vitro artifact. Also, high levels of methemoglobin (iron in the ferric state) have been found in spite of adequate levels of methemoglobin reductase (20). In most reptiles ATP is the primary regulator of oxygen affinity (21,22). In contrast, hemoglobin in crocodiles and alligators is unique (23), in that it is not responsive to organic phosphates but rather is regulated primarily by the bicarbonate ion which induces a decrease in oxygen affinity. The loss of sensitivity to phosphates apparently is caused by replacement of Pro or Ser for His at xcex2 NA2 and replacement by Ala for His at xcex2 H21. Also, the N-terminus of the chain of the hemoglobin from Alligator mississippiensis is blocked by an acetyl group (24,25,26). This alteration at the N-terminus permits hydrogen bonding with bicarbonate ions on the chains (25,26).
Mammalian hemoglobin-based compositions have been developed for administration as blood substitutes. (27,28) These include chemically modified hemoglobin which contains the oxygen-carrying heme group required for proper oxygen transport. While such modified hemoglobin-based compounds have been administered as blood substitutes, administration of unmodified hemoglobin, its heme free subunits or fragments or synthetic peptides therefrom has not previously been disclosed for this purpose or for other therapeutic uses. Indeed, the heme free xcex1 and xcex2 subunits would not be utilized for the purpose of providing blood substitutes, as they are incapable of binding oxygen.
The present invention provides a method for killing bacteria or fungi comprising contacting the bacteria or fungi with an antimicrobially effective amount of reptilian hemoglobin protein, hemoglobin protein fragment or polypeptide fragments thereof selected from the group consisting of intact hemoglobin, heme-free hemoglobin xcex1 chain, heme-free hemoglobin xcex2 chain, fragments of said proteins or polypeptide fragments thereof and combinations thereof.
The invention also provides a method for treating a subject having a bacterial or fungal infection comprising administering an antimicrobially effective amount of said protein, polypeptides and/or fragment compositions, to a method for treating material subject to bacterial or fungal contamination comprising applying to or admixing with said material an antimicrobially effective amount of said compositions, and to the use of said compositions for antimicrobial treatment of bacteria or fungi.
The invention additionally provides a pharmaceutical dosage form comprising an antimicrobially effective amount of said protein, polypeptide and/or fragment compositions and pharmaceutically acceptable carriers.